1. Field of the Invention
This invention generally relates to methods for forming dyed microspheres and populations of microspheres. Certain embodiments include attaching a hydrophilic dye to chemical groups to form a bubble and disposing the bubble and a microsphere to be dyed in a solvent such that the bubble is incorporated into the microsphere.
2. Description of the Related Art
The following descriptions and examples are not admitted to be prior art by virtue of their inclusion within this section.
Spectroscopic techniques are widely employed in the analysis of chemical and biological systems. Most often, these techniques involve measuring the absorption or emission of electromagnetic radiation by the material of interest. One such application is in the field of microarrays, which is a technology exploited by a large number of disciplines including the combinatorial chemistry and biological assay industries. One company, Luminex Corporation of Austin, Tex., has developed a system in which biological assays are performed on the surface of variously colored fluorescent microspheres. One example of such a system is illustrated in U.S. Pat. No. 5,981,180 to Chandler et al., which is incorporated by reference as if fully set forth herein. In such a fluid flow device, microspheres are interrogated by laser excitation and fluorescence detection of each individual microsphere as it passes at relatively high speed through a detection zone. Measurement data generated by such a system may be easily exported to a database for further analysis.
In the above-mentioned system, fluorescent dyes are absorbed into the microspheres and/or bound to the surface of the microspheres. The dyes are chosen based on their ability to emit light having a wavelength within a detection window of the measurement system. Further, the detection windows of the measurement system are spaced apart by a number of wavelengths, and the dyes are typically designed to minimize the overlap of a dye's fluorescent signal within adjacent detection windows. By employing two detection windows and two dyes, each at 10 different concentrations, there would thus be 100 fluorescently distinguishable microsphere sets.
Since dyes are primarily chosen for their fluorescence characteristics, sometimes dyes that have excellent fluorescent characteristics may be substantially incompatible with the microspheres to which they must be coupled. For example, one widely used dye is phycoerythrin (PE). This dye is hydrophilic. In contrast, the polymers of which microspheres are usually formed are hydrophobic. As such, it may be impossible to incorporate this dye into microspheres with currently used dyeing methods.
For example, one particularly suitable method for incorporating a dye into the polymer of microspheres includes disposing the microspheres in a solvent that causes swelling of the microspheres. When a dye is also disposed in the solvent, the dye will migrate into the polymer core of the microspheres. The dye can then be trapped in the polymer core by de-swelling the microspheres. De-swelling may be caused by changing the solvent.
Typically, the solvent that is used to swell the microspheres is an organic solvent or contains an organic solvent. In this manner, a hydrophilic dye may not be adequately dissolved, dispersed, or suspended in the solution that includes the microspheres and the solvent such that the microspheres can be dyed at all, let alone uniformly. If the hydrophilic dye can be sufficiently forced into the solution, the organic solvent would possibly destroy the hydrophilic dye. Therefore, hydrophilic dyes often cannot be incorporated into the polymer core of microspheres with currently used dye incorporation methods.
Other currently used methods for coupling hydrophilic dyes to the hydrophobic polymer core of microspheres include attaching a hydrophilic dye to an outer surface of the polymer core. Attaching the hydrophilic dye to the outer surface of the polymer core greatly reduces the complexity of the dyeing process. For example, such dyeing procedures can be carried out in aqueous solvents thereby eliminating the problems outlined above.
There are, however, several potential disadvantages to attaching the dye to the outer surface of the polymer core. For example, one or more characteristics of a surface bound dye may be affected by changes in the buffer solution in which the dyed microspheres are disposed, for example, during an assay. Such changes in the one or more characteristics of the dye due to the buffer solution may include changes in brightness (intensity) and color (wavelength). In addition, the shelf stability of a dye attached to the surface of the polymer core of microspheres may be less than the shelf stability of a dye incorporated into the polymer core. For example, if a dye can be incorporated in the polymer core, the dye may be better protected from buffer solutions and other conditions to which the microspheres are exposed. Furthermore, the variation in dyeing results are generally higher for surface attaching of a dye to a microsphere than for polymer core incorporation of the dye. In other words, microspheres that have dyes incorporated into the polymer core generally have a lower % coefficient of variation (% CV). A low % CV is advantageous for a number of reasons known in the art such as more uniform microsphere fluorescence emission characteristics and high microsphere classification accuracy.
Accordingly, it would be advantageous to develop a method for incorporating a hydrophilic dye into a polymer core of a microsphere without destroying the dye or otherwise adversely affecting characteristics of the dye such as intensity and wavelength of fluorescence emission to thereby produce a dyed microsphere having highly stable dye characteristics (e.g., a relatively long shelf life), with relatively high uniformity from microsphere-to-microsphere (or low % CV within microsphere subsets), and without increasing the difficulty or complexity of the actual dyeing process.